Alcohol potentiates RSV-mediated injury to ciliated airway epithelium

Abstract

Alcohol impairs resolution of respiratory viral infections. Numerous immune response pathways are altered in response to alcohol misuse, including alcohol-induced ciliary dysfunction in the lung. We hypothesized that mucociliary clearance-mediated innate immunity to respiratory syncytial virus (RSV) would be compromised by alcohol exposure. Cilia were assayed using Sisson-Ammons Video Analysis by quantitating the average number of motile points in multiple whole field measurements of mouse tracheal epithelial cells grown on an air-liquid interface. Pretreatment with ethanol alone (100 mM for 24 hours) had no effect on the number of motile cilia. A single dose (TCID₅₀ 1 × 10⁵) of RSV resulted in a significant (p < 0.05) decrease in motile cilia after 2 days. Ethanol pretreatment significantly (p < 0.05) potentiated RSV-induced cilia loss by 2 days. Combined RSV and ethanol treatment led to a sustained activation-induced auto-downregulation of PKC epsilon (PKCε). Ethanol-induced enhancement of ciliated cell detachment was confirmed by dynein ELISA and LDH activity from the supernates. RSV-induced cilia loss was evident until 7 days, when RSV-only infected cells demonstrated no significant cilia loss vs. control cells. However, cells pretreated with ethanol showed significant cilia loss until 10 days post-RSV infection. To address the functional significance of ethanol-enhanced cilia detachment, mice fed alcohol ad libitum (20% for 12 weeks) were infected once with RSV, and clearance was measured by plaque-forming assay from lung homogenates for up to 7 days. After 3 days, RSV plaque formation was no longer detected from the lungs of control mice, while significant (p < 0.01) RSV plaque-forming units were detected at 7 days in alcohol-fed mice. Alcohol-fed mice demonstrated enhanced cilia loss and delayed cilia recovery from tracheal measurements in wild-type C57BL/6 mice, but not PKCε KO mice. These data suggest that alcohol worsens RSV-mediated injury to ciliated epithelium in a PKCε-dependent manner.

Keywords: Ciliary beat frequency; Mouse tracheal epithelial cell; Protein kinase C; Respiratory syncytial virus.

Fig. 1.. The combination of alcohol and RSV potentiates cilia slowing coincident with PKC epsilon action in mouse tracheal epithelial cell cultures.

Ciliated mouse tracheal epithelial cells (MTEC) cultured at air-liquid interface and pretreated 24 hr with or without 100 mM alcohol (EtOH) were infected with 1 × 10⁵ TCID₅₀ respiratory syncytial virus (RSV) for 1–6 hr. Control cells received no treatment (Media). A) Ciliary beat frequency (CBF) decreased in RSV-treated MTEC (^ap<0.05 vs Media at matched time points of 3–6 hr) while EtOH+RSV further enhanced cilia slowing due to RSV only (^bp<0.05 vs RSV only at matched time points of 3–6 hr). EtOH+RSV potentiated the time of RSV-induced CBF decrease compared to media control (^cp<0.01 vs Media at 1 hr). B) Protein kinase C epsilon (PKCε) activity was increased by RSV (^ap<0.05 vs Media at 1 hr), enhanced by EtOH+RSV (^bp<0.01 vs Media at matched time points of 1–2 hr), and significantly decreased with EtOH+RSV (^cp<0.05 vs Media or RSV only at matched time points of 5–6 hr). Experiments were performed independently three times each using triplicate data measurements (n=9).

Fig. 2.. The combination of alcohol and RSV results in worsened cilia loss and cell death in mouse tracheal epithelial cell cultures.

RSV-induced damage to ciliated cells was quantitated by dynein ELISA and LDH release. A) RSV infection resulted in increased axonemal dynein in the culture media by 6 hr (^ap<0.05 vs Media) while 24 hr pretreatment with EtOH increased RSV-induced cilia loss (^bp<0.001 vs RSV only). B) RSV infection increased LDH release (^ap<0.005 vs Media) and this release was enhanced in EtOH pre-treated MTEC (^bp<0.02 vs RSV only). Experiments were performed independently three times each using triplicate data measurements (n=9).

Fig. 3.. Alcohol feeding in mice potentiates and sustains RSV-induced cilia slowing and cilia loss and delays recovery of the ciliated epithelium.

Female BALB/c mice were ethanol-fed or water-fed for 8 wks prior to RSV infection (3,250 TCID₅₀ U/mouse) and sacrificed 1–7 days post infection. A) CBF decreased in tracheal epithelium of RSV-infected mice (^ap<0.05 vs Control mice) by 24 hr and recovered to baseline levels by day 2. In alcohol- (EtOH) fed mice, the RSV-mediated CBF decrease persisted through 1 wk (^ap<0.05 vs Control and RSV only mice @1wk). B) RSV caused a loss of motile cilia (^bp<0.05 vs Control mice at 48 hr) which recovered by 1 wk after infection. In alcohol-fed RSV-infected mice, cilia loss was detected 24 hr earlier and sustained longer than RSV only (^ap<0.05 vs RSV only at 24 hr and 1 wk and Control mice at all time points). n=6–10 mice per group from replicated experiments.

Fig. 4.. Alcohol feeding in mice potentiates and sustains RSV-induced PKC epsilon auto-downregulation.

PKCε activity was assayed in the same mouse tracheal epithelial samples from in vivo-exposed mice shown in Fig.3. RSV infection decreased PKCε activity (^bp<0.05 vs Control mice at 24–48 hr), but returned to baseline by day 3–7. Decreased PKCε activity below baseline levels (autodownregulation) after RSV infection persisted in EtOH-fed mice (^ap<0.01 vs RSV only at 72 hr -1 wk and Control mice at all times). n=6–10 mice per group from replicated experiments.

Fig. 5.. Alcohol feeding in mice prevents normal clearance of RSV.

RSV titers were determined in lung homogenates (100 μg/mL) from 1–7 days post-infection in control- and alcohol-fed mice. RSV was equally detected in lung homogenates of control- and alcohol-fed mice at 24 hr post infection. RSV continued to be detected in the lungs of alcohol-fed mice out to 1 wk, compared to control-fed mice (^ap<0.05 vs RSV only mice at 2–7 d). n=6–10 mice per group from replicated experiments.

Fig. 6.. The combination of alcohol feeding and RSV infection does not slow or detach cilia in MTECs from PKCε knockout mice.

MTEC from wild type and PKCε KO mice were cultured under air-liquid interface conditions and treated with 1 × 10⁵ TCID₅₀ RSV in the presence of 100 mM EtOH. Baseline CBF and the number of motile points were preserved in PKCε KO MTEC (^ap<0.01 vs wild type or PKCδ KO mice) for both A) cilia slowing at 3 hr and B) number of motile points at 8 hr.

Fig. 7.

Model diagram for temporal regulation of cilia slowing by PKCε activation leading to subsequent ciliated cell detachment by PKCε auto-downregulation.